Lipase-Catalyzed Esterification Of Rutin And Physicochemical Properties Of Rutin Aliphatate

Flavonoids are widely distributed in different parts of miscellaneous plants and exhibit a wide range of biological activities, such as anticancer, anti-inflammatory, anti-virus, anticoagulant, anti-atherosclerosis, and inhibitory effects on low-density lipoprotein (LDL) oxidation. Due to their biological properties, the use of flavonoids in food, cosmetic and pharmaceutical preparations is currently the subject of great interest. Unfortunately, most of naturally occurring flavonoids (mainly flavonoid glycosides) show a low solubility and stability in the lipophilic media and ineffectiveness in stabilizing fats and oils. These properties limit the development of nutrition food and commodity containing flavonoids. Most of biological activities of flavonoids may arise from their resistance to oxidation including scavenging free radicals, chelating transition metal ions, and protecting the lipid from peroxidization. Aglucone show more excellent anti-oxidation compared with glucosides because the former exhibit strong lipotropy and partition preferentially into the hydrophobic core of the bio-membrane. These results are confirmed by protecting LDL and the complex of carotenoids and unsaturated fatty acids from oxidation by flavonoids. Generally speaking, the hydrophilic property of flavonoid glucosides, which dues to polyhydroxyl and saccharide groups, will compress their anti-oxidative activity. Esterification of the hydroxyl groups in molecules by fatty acids is a possible way to improve the hydrophilic property of flavonoid glucosides. In the classical esterification catalyzed by chemical catalyst, most of hydroxyl groups may be acylated, which leads to a mixture of products with various degree of esterification. Once the active hydroxyls are acylated, the biological activity of flavonoids will be reduced or even lost. However, the enzymatic catalysis is an alternative because enzymes are very regioselective. More attention has recently been paid to enzymatic esterification of flavonoid glucosides. By now a lot of literatures have reported the effect of water content, acyl donor structure, flavonoid backbone, enzyme origin and solvent nature on the results of the acylation. However, the effect of addition time of molecular sieves on the conversion in esterification has not yet been reported in some detail. The longest alkyl chain of acyl donor that has been reported by now is hexadecyl. The esterification by acyl donors with longer alkyl chains will bring rutin esters some new properties, such as amphipathy. Thus, in our experiments, rutin was acylated with a series aliphatic acid in esterification reaction catalyzed by immobilized Candida antarctica lipase B (Novozym 435) in tert-arnyl alcohol. The lipophilic rutin stearate was synthesized by this method. The structure of turin esters were characterized by spectra methods of ¹H-NMR and ¹³C-NMR, FT-IR and UV-Vis. The results suggested that the regioselectivity of the lipase-catalyzed esterification of rutin was specific at the C4'" position of the rhamnose moiety. It was found that the addition Of molecular sieves increased both of the reaction rate and the yield obviously. For the esterification which stearic acid was the acyl donor, the final conversion for the case to add molecular sieves at 24 h after the beginning of reaction was the highest, i. e. about 46%. And the highest conversion, i. e. about 64.5%, for the esterification which lauric acid was acyl donor was obtained with adding molecular sieves at 11 h. The UV-Vis spectrums of rutin esters, what have not been reported until now in other literatures, were very similar to that of rutin. The main peaks were 256 and 357 nm in the UV-Vis spectrum for rutin esters.The absorption and metabolism of pharmaceuticals involve the interaction of pharmaceuticals with bio-membrane, which leads to the changes of structure and physio-properties of bio-membrane, such as permeation, fluidity. Lipid monolayer is usually chosen as simple model of bio-membrane, so the investigation on interaction of flavonoids with lipid monolayer may be a simple as well as effective way to reveal the antioxidative activity mechanism of flavonoids. However, few papers have reported the interaction of flavonoids with lipid monolayer, more with the lipid bilayers. This probably because flavonoids can not spread at air/water interface, as other conjugated aromatic molecules. However, amphiphilic flavonoids esters which are obtained by esterification could spread at air/water interface to form monolayer. The properties of monolayers formation of amphiphilic flavonoid esters at the air/water is the precondition for investigating the interaction of flavonoids with lipid monolayer. However, little attention has by now been paid to this subject. In our experiments, we explored the monolayers formation of RS, RL and RC at the air/water interface, changing the compression rate. We also report the influences of Al³⁺ in subphase toÏ€-A isotherms of RS and RL monolayer. Although the introduction of alkyl chains makes the rutin, which does not spread at air/water interfaces, amphiphilic, the properties of monolayers depend on the alkyl chain length, cmpression rate and the subphase. Independent of subphase, the isotherms of RS and RL monolayers with liquid-phase are observed. Near to the collapse of monolayers, the aglucons of rutin esters lie on the surface with a tightly packing of glucose and rhamnose, which are oriented perpendicularly to the surface and point into the water phase, and alkyl chains point into air loosely. Both RS and RL have not enough hydrophobicity to form monolayers which are stable enough to maintain the surface pressure for a long period. RL with shorter alkyl chain shows a more pronounced tendency of dissolution. The molecular area of the RS and RL monolayers decreased as the compression rate was reduced, however, theÏ€_coll increased. RC was so soluble that it was not capable of forming monolayer at air/water interface. When RS, RL and RC were spread on aqueous aluminum solution, the values of a_coll andÏ€_coll of monolayers are larger compared to spread at air/water interface, and RC could spread to form monolayer with liquid-phase.Although amphipathic rutin esters could spread at water/air interface, they were soluble in water a little. The surface properties of rutin esters adsorbed at the water/air interface from bulk phase could be the supplement and comparison for that of spread monolayer of rutin esters. However, the adsorption of rutin esters at water/air from bulk phase has not been reported by now. In our experiments, the surface properties of rutin esters adsorbed at the surface of diluted alkali solution. The reason why choose diluted alkali solution was that the solubility of rutin esters in water was so low that 1 mg rutin esters could not dissolve in 1000 ml water, while rutin esters could dissolve in alkali solution. But rutin would ionize and form sodium salt in the alkali solution if pH＞8.77, thus we chose diluted alkali solution, of which the value of pH was about 8, as experiment solution. The measurement of surface pressure of rutin esters in diluted alkali solution was performed using a KSV instrument of interfacial tension with a Wilhelmy type microbalance using a platinum plate. The suface properties of rutin esters adsorbed at the surface were investigated. The surface pressure of RS, RL and RC decreased with the increasing of their concentration. Surface activity was more effective and the value of CMC was higher with longer alkyl chain of rutin esters. The values of d_γ/dlgC of all rutin esters were negative, which indicated the positive adsorption. Respectively, the maximum of surface excess of RS, RL and RC were 1.71×10^-11 mol/cm²,1.35×10^-11 mol/cm² and 1.41×10^-11 mol/cm² and the concentration of which were 3×10^-3 mg/mL,1.65×10^-3 mg/mL and 1.3×10^-3 mg/mL. The adsorption isotherm of the rutin esters were all Langmuir adsorption. At standard state, they all could adsorb at surface spontaneously.Stearic acid is usually mixed with other amphiphilic molecules to study the two-component mixed monolayer. In the present paper, the characteristic such as miscibility, thermodynamic stability and compressibility of RS/SA and RL/SA mixed monolayers have been investigated by Langmuir film balance to yield quantitative information on the nature of the molecular interaction. These are the precondition for investigating the interaction of flavonoids with lipid monolayer. The collapse pressures of the mixed monolayers increase gradually with increasing proportion of stearic acid and are between that of the pure components, which mean RS/SA and RL/SA monolayers are miscible throughout the mixture composition range. However, with the increasing of X_SA, the attraction between molecules of the two components in RS/SA mixed monolayers turns to be repulsion and the miscibility of two components turns to be bad. At X_SA=0.14-0.16 the prominent negative deviations from ideal molecular areas and the most negative values ofâ–³G_ex are observed with an exception for the case of 25 mN/m, where the maximum deviations and the most negative excess free energies are observed at X_SA=0.25. The deviations from ideal molecular areas and the excess free energies of RL/SA are all positive at any mole fraction of stearic acid. The miscibility and stability of RS/SA mixed monolayers are better than those of RL/SA mixed monoalyers. This probably because RS has longer alkyl chains and favors the array of hydrophobic groups of two components of mixture. The compressibility of the mixed monolayers seems to be sensitive to both the amount of stearic acid component and the surface pressure. The considerable fluctuations of comressibility of RS/SA and RL/SA mixed monolayers with the various X_SA were observed and the greatest compressibility were both obtained atÏ€=5 mN/m.Monolayers of dioleoylphosphatidylcholine (DOPC) are used as model membranes to study their molecular interaction with RS and RL. The characteristic such as miscibility, thermodynamic stability and compressibility of DOPC/RS and DOPC/RL mixed monolayers have been investigated by Langmuir film balance to yield quantitative information on the nature of the molecular interaction, which can provide valuable insights to reveal the antioxidative activity mechanism of flavonoids. The effects of temperature of the subphase on the DOPC-rutin esters interactions are investigated. Pure RS, RL and DOPC were all capable of forming monolayers with liquid-phase at the air/water interface. The two components of DOPC/RS and DOPC/RL monolayers were miscible throughout the mixture composition range. The miscibility was more excellent at higher temperature. At different temperatures, the condensing effect of RS on the DOPC monolayer, which represented the attractive forces between DOPC and RS, is more significant at lower surface pressures. This condensing effect could be ascribed to the match of molecular shapes of RS and DOPC on the monolayers packing. The less miscibility of RL and DOPC may be dues to its shorter alkyl chain. At some mole fraction of RS and surface pressure, DOPC and RL molecular shapes may mismatch, and the cohesion between RL and RL, DOPC and DOPC is greater than RL and DOPC. But high temperature and low X_RL both could improve the miscibility of RL and DOPC. The values ofâ–³G_ex of RS/DOPC monolayers are all negative at any X_RS and surface pressures, with the exception for the case ofÏ€= 30mN/m, X_RS=0.75, whereâ–³G_ex is positive. In the range of 0.33＜X_RS＜0.47 for all surface pressure studied, the mixture exhibits the greatest thermodynamic stability compared with the pure component monolayers. The two components of DOPC/RL monolayers mix spontaneously only for X_RL＞0.5 andπ＜25 mN/m. At any temperatures studied, the compressibility of DOPC/RS monolayers increased with the increasing of X_RS and the decreasing of the surface pressure. This phenomenon was also observed for DOPC/RL monolayers.Rutin is famous and typical one of flavonoids. Esterification of the hydroxyl groups in molecules by aliphatic acid is a possible way to improve the hydrophilic property of rutin. We expect that rutin esters will inset into bio-membrane with alkyl chains locating at the hydrophobic core. The hydrophobic interaction between the alkyl chains steadies the location of rutin esters in the bio-membrane. Furthermore, the anti-oxidation groups keep retentive during the esterification of rutin. Thus, more prominent antioxidative activity may be expected for rutin esters. However, the investigations about the antioxidation of rutin esters have been reported by now. In our experiments, using the content of thiobabituric acid reactive substance (TBARS) as index, the inhibitory effects of rutin and rutin esters on oxidative modification of lecithin induced by Fe₂₊ were compared. The results showed that, rutin and rutin esters could inhibit the oxidative modification and the role of rutin esters was better than that of rutin; rutin caporate, which has moderate solubility in water, was the best in three kinds of rutin esters.Rutin is adsorbed and metabolized via body fluid in vivo, while its three aliphatic esters, i. e. stearic ester, lauric ester and caproic ester, are weak soluble in water. In our experiments, rutin laurie ester was chosen as sample to prepare solid dispersions for increasing its water solubility. PEG4000 was taken as a carrier, and solid dispersions of rutin laurie ester with different ratio were prepared by methanol solution method. The solubility of the solid dispersion was significantly increased, compared with the rutin laurie eater, and its solubility increased with the increase of content of PEG4000. IR spectrophotometer and UV spectrophotometer were used to investigate the solid dispersion. It is clear that there was no chemical reaction between rutin laurie ester and PEG4000.